In recent years, a massive amount of genes, proteins, and the like have been enabled to be comprehensively analyzed according to development in technologies such as microarray or macroarray experiments. For example, in a DNA microarray, several hundreds to several tens of thousands of DNAs may be arrayed in a matrix to be fixed as spots on a carrier such as a slide glass, and mRNA or cDNA extracted and labeled from a cell to be examined may be hybridized with the spots to measure a gene expression level.
That is, a substance to be examined such as labeled cDNA selectively binds to a complementary DNA on the carrier so that the gene expression level can be estimated by acquiring detection intensity of a label. Although reliability as data is required for a selective binding amount of the substance to be examined such as the gene expression level, the detection intensity may vary depending on non-biological factors such as when selective binding substances such as complementary DNAs fixed as spots on the carrier are unevenly distributed or a case in which dust is attached to a spot portion.
Accordingly, a method has been developed to determine uniformity of spots in a DNA microarray and the like. For example, a method of evaluating the uniformity disclosed in Japanese Patent Application Laid-open No. 2004-340574 includes the following steps: (1) background data corresponding to each of the spots is obtained by adapting analyzing software to an image obtained by scanning a monochromatic light emitting image of the DNA microarray, (2) plate-to-plate No. and a plate position of a target DNA corresponding to each of the spots are calculated, (3) the plate-to-plate No. and the plate position are allowed to correspond to each piece of the background data, and (4) the pieces of background data are arranged in the order of the plate No. and the plate position to obtain a sequence BG and a periodicity rule is detected by extracting a sub-sequence from the entire sequence.
Japanese Patent Application Laid-open No. 2008-039584 discloses that unevenness is evaluated with reference to a value of a coefficient of variation (CV). Therein, the CV of the spot means a proportion (%) of a standard deviation (SD) to an average value of fluorescence intensity of each spot that is obtained when the detection intensity of a spot of the DNA microarray and the like on the carrier is scanned and measured.
Regarding an analyzing software of microarray GenePix Pro (manufactured by Molecular Devices, LLC.), each intensity value of pixels included in the spot is compared to an average intensity value of pixels around the spot, and the spot is determined to be defective if a predetermined proportion of the pixels included in the spot satisfies the condition as follows: “(intensity of each pixel included in the spot)−(average intensity value of the pixels around the spot)<0” (Inter Medical Co., Ltd., “GenePix Pro 7.0 Microarray Image Analysis,” [online], Copyright 2006 Inter Medical Co., Ltd., [retrieved on Mar. 6, 2012]. According to this, it is detected that a background around the spot becomes high due to certain abnormality, for example, dust is attached thereto or a chip is contaminated.
However, reliability of the selective binding amount of the substance to be examined cannot be appropriately determined by the method of determining the uniformity of spots in the art.
Specifically, although the uniformity among a plurality of spots can be evaluated by the method of evaluating the uniformity disclosed in JP '574, uniformity within single spot cannot be evaluated.
In the evaluating method with the CV value disclosed in JP '584, when intensity of some pixels in a pixel group configuring the spot is extremely high or low due to attachment of dust or the like, the standard deviation becomes large and the CV value exceeds a threshold. Accordingly, even though intensity data of the pixel group except for some pixels can be used, the data is eliminated, which causes excessive detection.
That is, in general, a median (median value) of the pixel group within the spot in an image is used as a representative value of signal intensity of a spot on a DNA chip and the like. For example, when alignment is performed in a circular spot having a diameter of 100 micrometers in the image, a median (spot median) of intensity is obtained for a pixel group of about 70 pixels (pixel size: 10 micrometers square) included in the circle.
This is because the median hardly varies depending on outliers as compared to using an average value of intensity of the pixel group. That is, when there are outliers such as an extremely large value and an extremely small value of the detection intensity of the pixel group in the spot, the overall average is skewed by the outliers.
FIG. 1 is a scatter plot illustrating detection intensity when the same substance to be examined is hybridized to two DNA chips. The horizontal axis represents the detection intensity of each spot in one of the DNA chips, and the vertical axis represents the detection intensity of each spot in the other DNA chip. That is, coordinates (X, Y) of one point represents detection intensity (X) measured in one DNA chip and detection intensity (Y) measured in the other DNA chip for the spot to which the same selective binding substance is fixed.
In that example, the same substance to be examined is hybridized to the two DNA chips so that ideally Y=X should be satisfied. However, as illustrated in FIG. 1, intensity plots of spots 1 and 2 are largely deviated from a line of Y=X so that it is considered that there is a defective spot. FIG. 2 is a diagram illustrating an intensity image of the spot 1 (left figure) and a variation graph of the detection intensity along the longitudinal line of the spot 1 (right figure). FIG. 3 is a diagram illustrating an intensity image of the spot 2 (left figure) and a variation graph of the detection intensity along the longitudinal line of the spot 2 (right figure). In the intensity image, a dashed-line circle indicates a spot portion, and the detection intensity is represented with a white gradation value.
As illustrated in the left figures of FIG. 2 and FIG. 3, the detection intensity is not represented as a uniform circle, but as a non-uniform gradation, in the intensity image of the spot portion. The variation graphs (right figures) represent a variation of the detection intensity along the longitudinal line of the spot portion in a range of one pixel in the horizontal direction and more than ten pixels in the longitudinal direction. Ideally, the detection intensity should be constant, but it largely varies. A main cause of the defect is a defective spot (a defect caused when the selective binding substance is fixed) and attachment of dust.
In the related art such as disclosed in JP '584, to eliminate such a defective spot, quality of the spot is determined using the CV value obtained as follows:CV value=(standard deviation of intensity of pixel group)/(average value of intensity of pixel group).
That is, the data has been eliminated as a defective spot when the CV value is equal to or larger than a predetermined reference value. In that case, when 5 pixels in the pixel group including 70 pixels indicate extremely large values in the above example because dust and the like is attached to part of the spot, the CV value becomes equal to or larger than the reference value and the data is eliminated.
However, the median of the pixel group in the spot is usually used as the representative value of the signal intensity of the spot so that the value hardly varies even when the outlier is included in part of the spot. Accordingly, the data may be sufficiently usable. That is, in the conventional evaluating method using the CV value, sufficiently usable data has been eliminated by excessive detection of defective spot.
It could therefore be helpful to provide a determination method, a determination device, a determination system, and a computer program that can appropriately evaluate reliability of a selective binding amount of a substance to be examined considering non-biological effect for data obtained from a microarray experiment and the like.